Indigenous intestinal bacteria play a vital role within the human body, and are important for host functions related to cancer risk, including the production and/or metabolism of carcinogens and maintenance of immune function. Marked interindividual differences in intestinal bacteria exist in humans, which may ultimately translate to differences in cancer susceptibility. For example, equol production is a biomarker of variability in indigenous microflora; 30-50% of the population harbor bacteria that metabolize the soy isoflavone daidzein to equol. Several studies suggest that equol production may be associated with a reduced risk of breast and prostate cancers. Many (40-60%) intestinal bacteria cannot be cultured ex vivo, but several molecular methods are now available that utilize bacterial RNA and DMA to quantitate bacterial species and characterize baterial communities regardless of whether or not the bacteria can be cultured. The utility of these methods in population-based human studies has not been established. We propose to develop a quantitative, high-throughput method to characterize human intestinal microbial communities. The method will combine real-time quantitative PCR (QPCR) with terminal restriction length polymorphism (tRFLP) of the baterial 16S rRNA gene to provide the quantitative foundation for analysis of fecal microbial communities. We propose to test this methodology in several ways: we will establish and validate a robust method for fecal sample collection and storage; we will assess within- and between-individual variability in fecal microbial communities; and we will compare fecal microflora in samples from known equol-producers and non-producers. Stored fecal samples are available for assay development, assessment of between-individual variability, and comparison of equol-producers and non-producers. For evaluation of sample-handling and within-individual variability, 10 healthy volunteers will be recruited to provide a fresh fecal sample on four occasions over three months. The proposed study will provide the groundwork, in an efficient and cost effective manner, for future studies to assess relationships between human intestinal bacteria and cancer risk, utilizing molecular techniques for the assessment of human intestinal microbial populations.